Journal of Molecular Biology
Regular articleActive and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism1
Introduction
Aerobic organisms benefit substantially from the high energy yields obtained via controlled conversion of molecular oxygen to water, yet reactive intermediates are a burden that cause cellular damage. Catalase protects hemoglobin by removing over half of the hydrogen peroxide generated in normal human erythrocytes, which are exposed to substantial oxygen concentrations (Gaetani et al., 1989). Catalase has been implicated as an important factor in inflammation (Halliwell & Gutteridge, 1984), mutagenesis (Vuillame, 1987), prevention of apoptosis Yabuki et al 1999, Islam et al 1997, Sandstrom and Buttke 1993, and stimulation of a wide spectrum of tumors (Miyamoto et al., 1996). Loss of catalase leads to the human genetic disease known as acatalasemia, or Takahara’s disease (Ogata, 1991). Intriguingly, mutations in a cytosolic Caenorhabditis elegans catalase shorten adult life span (Taub et al., 1999), supporting a role for catalase and reactive oxygen species in modulating the aging process in eukaryotes. In the brain, the reaction of ethanol with catalase is an important source of acetaldehyde Zimatkin et al 1998, Hamby-Mason et al 1997, which is implicated in the neurological effects of alcohol in humans (Hunt, 1996). Treatment of rats with the catalase inhibitor, 3-amino-1,2,4-triazole (3AT), decreases voluntary ethanol consumption (Aragon & Amit, 1992).
Heme-containing catalases have been identified in organisms from bacteria to humans. They convert two molecules of hydrogen peroxide to two molecules of water and one molecule of oxygen (von Ossowski et al., 1993). The catalytic mechanism is a two-step reaction (Deisseroth & Dounce, 1970). In the first step, the heme Fe3+reduces a hydrogen peroxide molecule to water and generates a covalent Fe4+=O oxyferryl species with a porphyrin π-cation radical (Ivancich et al., 1997), referred to as compound I. In the second step, compound I oxidizes a second peroxide molecule to molecular oxygen and releases the ferryl oxygen species as water. Organic peroxides, such as peroxyacetic acid (PAA), can substitute for hydrogen peroxide by slowly forming compound I; however, they do not reduce catalase back to the resting state Chance 1949a, Chance 1949b.
With appropriate substrates, compound I can be reduced by a single electron to form compound II, in which the porphyrin radical is reduced, but the oxyferryl metal center is retained. Recent EPR experiments indicate that a species with a compound II-like absorption spectrum is formed by bovine liver catalase (BLC) compound I via electron transfer of the porphyrin radical to a tyrosine distant from the active site Ivancich et al 1996, Ivancich et al 1997. At least one or both of these oxidized states are unreactive to hydrogen peroxide (Chance, 1950) and can be generated during steady-state turnover of the enzyme Hillar et al 1994, Kirkman et al 1999.
Many heme catalases bind the reductant NADPH (Fita & Rossmann, 1985), yet hydrogen peroxide is the source of both oxidative and reductive potential during the normal catalytic cycle. The exact mechanism of NADPH action is unclear, although low catalase activity in xeroderma pigmentosum fibroblasts and SV40-transformed human cell lines are linked to low intracellular NADPH concentrations (Hoffschir et al., 1998). Recent experiments suggest that NADPH does not directly reduce trapped, off-pathway oxidized enzyme states, but rather prevents these states from forming in the first place by providing a more attractive source of reductant (Kirkman et al., 1999).
Human catalase is a central enzyme in the defense against oxidative damage and inactivation of hemoglobin in erythrocytes and relies upon a heme-dependent catalytic cycle, yet the only mammalian catalase structure solved to date is for BLC (Murthy et al., 1981) in which 50% of the heme is degraded. In order to address the human cell biology of reactive oxygen control, understand the mechanism of reaction and of inhibition, and to provide a molecular basis for understanding ethanol intoxication and human polymorphisms underlying acatalasemia in the Hungarian and Swiss populations and the 41 uncharacterized families of acatalasemia in the Japanese population (Goth, 1997), we defined the chemistry of the human catalase with structures of the resting-state enzyme and complexes of a catalase bound to the cyanide and 3AT inhibitors.
Section snippets
Structure determination and quality
We determined the crystal structure of human erythrocyte catalase in the space group P212121 with a biological tetramer in the asymmetric unit by using molecular replacement with BLC (Murthy et al., 1981) as a search model. The structure was refined to 2.2 Å resolution with an Rcryst of 17.2% and an Rfree of 22.7%. The electron density is clear for residues from Arg5 to Asn501 (Figure 1), and all observed residues possess excellent main-chain geometry with the exception of the conserved
Crystallization and X-ray diffraction data collection
Human erythrocyte catalase (EC 1.11.16, hydrogen peroxide:hydrogen peroxide oxidoreductase) was purchased from Calbiochem and concentrated to ∼40 mg/ml in 50 mM Tris (pH 8.0) for crystallization using vapor diffusion against 6.5%-8.0% PEG 4000. Data were collected on orthorhombic crystals that were cryogenically frozen in a cooled nitrogen stream after soaking mother liquor supplemented by 15% 2-methyl-2,4-pentanediol as cryoprotectant. Data from different frozen crystals were frequently
Acknowledgements
We thank D. B. Goodin, P. A. Williams, D. P. Barondeau, B. R. Crane, D. S. Daniels, S. S. Parikh, C. M. Bruns and C. D. Mol for generous and insightful discussion. In addition, we acknowledge our gratitude to M. E. Pique for assistance in generating Figure 3(a). We thank the staff at SSRL for assistance in data collection. This work was supported by the National Institutes of Health grant GM39345 to J.A.T. and a Howard Hughes Predoctoral Fellowship to C.D.P.
References (66)
- et al.
Simulations of electron transfer in the NADPH-bound catalase from Proteus mirabilis PR
Biochim. Biophys. Acta
(1995) - et al.
Crystal struture of catalase HPII from Escherichia coli
Structure
(1995) The primary and secondary compounds of catalase and methyl or ethyl hydrogen peroxide. II Kinetics and activity
J. Biol. Chem
(1949)The primary and secondary compounds of catalase and methyl or ethyl hydrogen peroxide. IV Reactions with hydrogen peroxide
J. Biol. Chem
(1949)- et al.
Laue diffraction study on the structure of cytochrome c peroxidase compound I
Structure
(1994) - et al.
Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes
Blood
(1989) - et al.
Crystal structure of Proteus mirabilis PR catalase with and without bound NADPH
J. Mol. Biol
(1995) - et al.
A novel human catalase mutation (358T→del) causing Japanese-type acatalasemia
Blood Cells Mol. Dis
(1995) - et al.
Low catalase activity in Xeroderma pigmentosum fibroblasts and SV-40 transformed human cell lines is directly related to decreased levels of intracellular levels of the cofactor, NADPH
Free Radical Biol. Med
(1998) Role of acetaldehyde in the actions of ethanol on the brain-a review
Alcohol
(1996)
TGF-β1 triggers oxidative modifications and enhances apoptosis in HIT cells through accumulation of reactive oxygen species by suppression of catalase and glutathione peroxidase
Free Radical Biol. Med
The function of catalase-bound NADPH
J. Biol. Chem
Mechanisms of protection of catalase by NADPH
J. Biol. Chem
Reaction of E. coli catalase HPII with cyanide as ligand and as inhibitor
Biochim. Biophys. Acta
Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli
J. Biol. Chem
Structure of catalase-A from Saccharomyces cerevisiae
J. Mol. Biol
XtalView/Xfit-a versatile program for manipulating atomic coordinates and electron density
J. Struct. Biol
Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 Å resolution
FEBS Letters
Structure of beef liver catalase
J. Mol. Biol
Transcriptional fidelity and proofreading by RNA polymerase II
Cell
Three-dimensional structure of catalase from Penicillium vitale at 2.0 Å resolution
J. Mol. Biol
Induced changes in the electron paramagnetic resonance spectra of mammalian catalases
Biochim. Biophys. Acta
Resistance to nitric oxide-mediated apoptosis in the HL-60 variant cells is associated with increased activities of Cu,Zn-superoxide dismutases and catalase
Free Radical Biol. Med
Roles of proximal ligand in heme proteinsreplacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase and catalase
Biochemistry
Heterogeneity of erythrocyte catalase IIisolation and characterization of normal and variant erythrocyte catalase and their subunits
Eur. J. Biochem
The effect of 3-amino-1,2,4-triazole on voluntary ethanol consumptionevidence for brain catalase involvement in the mechanism of action
Neuropharmacology
Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxina comparison with NAD bound to the oxidoreductase enzymes
Protein Sci
3D domain swappinga mechanism for oligomer assembly
Protein Sci
X-PLORVersion 3.1: A System for X-ray Crystallography and NMR Yale
The reaction of catalase and cyanide
J. Biol. Chem
The reactions of catalase in the presense of the notatin system
Biochem. J
Dinucleotide-binding site of bovine liver catalase mimics a catalase mRNA-binding protein domain
Am. J. Physiol
Solvent-accessible surfaces of proteins and nucleic acids
Science
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